Formation of the craniofacial skeleton appears to be far more complex than previously believed. For example, although both synchondroses (primary cartilage) and sutures and articulation (secondary cartilage) undergo endochondral ossification, they have different histological and biochemical profiles and different intrinsic growth potentials. In addition, although calvarial and facial bones appear to form by intramembranous ossification without cartilaginous primordia, we and other have observed transient expression of some cartilage-characteristic collagen genes during intramembranous ossification. The cellular and molecular mechanisms involved in the formation of primary and secondary cartilages and membranous bones of the craniofacial skeleton have not been elucidated. Collagens are crucial structural elements which determine the physicochemical properties of cartilage and bone. In addition, the genes encoding types I, II and III collagen may have additional functions in chondrogenesis that are unrelated to their roles in production of structural proteins. We have isolated two forms of a1(II) collagen mRNA produced by alternative splicing of the second exon; which encodes a cysteine-rich globular domain in the amino-propeptide. These two forms of alpha1(II) collagen mRNA are differentially expressed during chondrogenesis. In addition, alternative alpha2(I) and alpha1(III) collagen transcripts have been identified in cartilage. The cartilage a2(I) collagen mRNA is generated by transcription initiation from an alternative promotor, and the conceptual translation product of the mRNA is completely noncollagenous, indication from an alternative promotor, and the conceptual translation product of the mRNA is completely noncollagenous, indicating that it must have an alternative function in cartilage. The structure of the cartilage-specific type III collagen mRNA has not been completely determined. However, it is much too small to encode type III collagen; thus it may also have an alternative function in cartilage. In this application, we propose to use in situ hybridization and RNase protection assays to characterize the developing chick craniofacial skeleton in terms of the collagen mRNAs known to be important for chondrogenesis and osteogenesis, including the alternative mRNAs of the type I, II, and III collagen genes. We will compare the pattern of collagen gene expression in synchondroses (primary cartilage) and sutures and articulations (secondary cartilage) with that in the epiphyseal growth plate, which has been extensively characterized at the molecular and biochemical levels. We will also identify additional cartilage- characteristic collagen genes which are transiently expressed in differentiating calvaria (membranous bones). These experiments will permit us to further define the molecular mechanisms involved in endochondral ossification in primary and secondary cartilages and intramembranous ossification. More importantly, collective data from this project will form the foundation for a subsequent proposal regarding: 1) the functional roles of alternative type I, II and III collagen transcripts during normal and abnormal development of the craniofacial skeleton; and 2) the role of cartilage-characteristic collagen genes in intramembranous ossification.